The statistical optimization of fermentation medium components has revealed yeast extract, NaCl, peptone and K2HPO4 as the significant components. In this section, an attempt is made to identify their influence on the fermentative hydrogen production process. Yeast extract is a good complex nutritional source of amino-nitrogen, vitamins, and other unknown growth factors for many microbial organisms (Smith et al., 1975).
The main functional components in yeast extract (Johnson et al., 1981) that are used by Clostridia are p-aminobenzoic acid, vitamin B12, pyridoxamine, and biotin. Yeast extract is also necessary for the growth of the hydrogen producer C. thermopalmarium (Sohet al., 1991). Zhang et al., 2014 explained the importance of yeast extract on pH of the medium which is an important process parameter for fermentative hydrogen production and also affirmed that the yeast extract of high concentration (more than 2.0 g/L) might promote the role of hydrogen consumption at the latter part of the fermentation by activation of uptake hydrogenase. In this study, the optimized yeast extract value 1.48 g/L falls in this range and hence has a significant effect on H2 production by C.
pasteurianum. Similar studies were reported which also supported the significance of yeast extract on hydrogen production by clostridia (Geng et al., 2010; Ferchichi et al., 2005). Further, Li et al. (2011) have demonstrated that three amino acids (glycine, serine and tyrosine) in yeast extract accelerate the production of alcohol–dependent enzymes such as glycerol dehydrogenase, the immediate enzyme in glycerol metabolism. Peptone is another source of nitrogen which is required for cell growth and maintenance.
Beneficial effect of NaCl has already been supported in our previous work (Sarma et.al., 2016) as Na+ ions work towards faster trans-membrane transport of
nutrients and substrate to intracellular region, which essentially improves enzymatic reactions in glycerol metabolism resulting in higher hydrogen production. Xiaolong et al.
(2006)have studied the effect of sodium ion concentration on hydrogen production from sucrose and the optimum sodium ion concentration reported is in the range of 1–2 g/L.
Xiaolong et al. (2006)have also reported that Na+ concentrations above 4 g/L showed marked reduction in hydrogen production. This reduction is attributed to adverse effect of high Na+ concentration on microbial enzyme activities such as dehydrogenase activity, alkaline phosphatase and adenosine tri phosphate. Khanna et al. (2011) have assessed the effect of sodium ions on hydrogen production and concentration upto 250mM of sodium ion is beneficial for growth of hydrogen producing bacteria.
K2HPO4 in the medium acts as a source of both potassium and phosphate which are important growth enhancing nutrients. Moreover, K2HPO4 also acts as a buffering agent and it helps in maintenance of pH of the fermentation broth at a desired value (Yalcin and Ozbas, 2008) and pH is a significant factor for biohydrogen production process (Sarma et al. 2016).
4.4 CONCLUSIONS
This study attempted to optimize hydrogen production from crude glycerol using Clostridium pasteurianum. Hydrogen production was revealed to be influenced by medium components that augment activity and substrate promiscuity of hydrogenase and other enzymes involved in glycerol metabolism. Peptone is a source of nitrogen which is required for cell growth and maintenance, whereas K2HPO4 help maintain pH of the medium which is very essential for production of fermentative hydrogen. Yeast extract,
CHAPTER 4
which provides growth factors and amino acids accelerating production of alcohol degrading enzyme, was also revealed as essential component of the medium.
References
Chen, W.M., Tseng, Z.J., Lee, K.S., Chang, J.S., 2005. Fermentative hydrogen production with Clostridium butyricum CGSS isolated from anaerobic sewage sludge. Int J Hydrogen Energ. 30, 1063-1070.
Ferchichi, M., Crabbe, E., Hintz, W., Gil, G-H., Almadidy, A., 2005. Influence of culture parameterson biological hydrogen production by Clostridium saccharoperbutyl- acetonicum ATCC 27021. World J Microbiol. Biotechnol. 21, 855-862.
Geng, A., He, Y., Qian, C., Yan, X., Zhou, Z., 2010. Effect of key factors on hydrogen production from cellulose in a co-culture of Clostridium thermocellum and Clostridium thermopalmarium. Bioresour. Technol. 101, 4029–4033.
Jitrwung, R., Yargeau, V., 2011. Optimization of media composition for the production of biohydrogen from waste glycerol. Int. J. Hydrogen Energ. 36, 9602–9611.
Johnson, M.J., Peterson, W.H., Fred, E.B., 1931. Oxidation and reduction relations between substrate and products in the acetone butyl alcohol fermentation. J. Biol.
Chem.91, 569–591.
Junghare, M., Subudhi, S., Lal, B., 2012. Improvement of hydrogen production under decreased partial pressure by newly isolated alkaline tolerant anaerobe, Clostridium butyricum TM-9A: Optimization of process parameters. Int J Hydrogen Energy. 37, 3160-68.
Khamtib, S., Plangklang, P., Reungsang, A., 2011. Optimization of fermentative
hydrogen production from hydrolysate of microwave assisted sulfuric acid pretreated oil palm trunk by hot spring enriched culture. Int J Hydrogen Energ.
36,14204-14216.
Khanna, N., Kotay, S.M., Gilbert, J.J., Das, D., 2011. Improvement of biohydrogen production by Enterobacter cloacae IIT–BT 08 under regulated pH. J.
Biotechnol. 152, 9–15.
Khanna, S., Goyal, A., Moholkar, V.S., 2013a. Effect of fermentation parameters on bio–
alcohols production from glycerol using immobilized Clostridium pasteurianum:
an optimization study. Prep. Biochem. Biotechnol. 43, 828–847.
Li, M., Liao, X., Zhang, D., Du, G., Chen, J., 2011. Yeast extract promotes cell growth and induces production of polyvinyl alcohol–degrading enzymes. Enzyme Res.
2011, 179819. doi:10.4061/2011/179819.
Prasertsan, P., O-Thong, S., O-Thong, N.K., 2009. Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process. Int J Hydrogen Energ. 34, 7448-7459.
Plackett, R.L., Burman, J.P., 1946. The design of optimum multifactorial experiments.
Biometrika. 33, 305–325.
Ravikumar, K., Pakshirajan, K., Swaminathan, T., Balu, K., 2005. Optimization of batch process parameters using response surface methodology for dye removal by a novel adsorbent. Chem. Eng. J. 105, 131–138.
CHAPTER 4
Saraphirom, P., Reungsang, A., 2010. Optimization of biohydrogen production from sweet sorghum syrup using statistical methods. Int J Hydrogen Energ. 35(24), 13435-13444.
Sarma, S., Dubey, V.K., Moholkar, V.S., 2016. Kinetic and thermodynamic analysis (with statistical optimization) of hydrogen production from crude glycerol using Clostridium pasteurianum. Int. J. Hydrogen Energ. 33, 1471–1482.
Sittijunda, S., Reungsang, A., 2012. Biohydrogen production from waste glycerol and sludge by anaerobic mixed cultures. Int. J. Hydrogen Energ. 37, 13789–13796.
Smith, T.J., Hillier, A.J. and Lee, G.J. (1975) The nature of thestimulation of the growth of Streptococcus lactis by yeast extract.Journal of Dairy Research 42, 123–138.
Soh, A. L. L., Ralambotiana, H., Ollivier, B., Prensier, G., Tine, E. & Garcia, J.-L.
(1991). Clostridium thermopalmarium sp. nov., amoderately thermophilic butyrate-producing bacterium isolatedfrom palm wine in Senegal. Syst Appl Microbiol 14,135±139.
Tanyildizi, M.S., Ozer, D., Elibol, M.,2005. Optimization of α–amylase production by Bacillus sp. using response surface methodology. Process. Biochem. 40, 2291–
2296.
Wang, J., Wan, W., 2008. Optimization of fermentative hydrogen production process by response surface methodology. Int. J. Hydrogen Energ. 33, 6976–6984.
Xiaolong, H., Minghua, Z., Hanqing, Y., Qinqin, S., Lecheng, L., 2006. Effect of sodium ion concentration on hydrogen production from sucrose by anaerobic hydrogen–
producing granular sludge. Chinese J. Chem. Eng. 14, 511–517.
Yalcin, S.K., Ozbas, Z.Y., 2008. Effects of ammonium sulfate concentration on growth and glycerol production kinetics of two endogenic wine yeast strains. Indian
J.Biotechnol. 7, 89-93.
Zhang, L., Ban, Q., Li, J., Xu, Y. 2014. Assessment of Effects of Yeast Extract on Bio- hydrogen Production from Anaerobic Activated Sludge. Int J Agricult. Biol. 6, 1189-1193
CHAPTER 4
Chapter 5
Genetic Engineering of Clostridium pasteurianum for Improved Hydrogen Production
from Crude Glycerol
5.1 INTRODUCTION
Clostridium pasteurianum is a mesophilic, strictly anaerobic, Gram-positive bacterium that possesses the metabolic capacity to ferment glycerol as a sole source of carbon and energy, yielding a mixture of gases (hydrogen and carbon dioxide), acids (acetic and butyric), and alcohols (ethanol, butanol, and 1,3-propanediol) (Biebl et al., 2001 and Dabrock et al., 1992). Unlike the past industrial workhorses, C. acetobutylicum and C. beijerinckii; C. pasteurianum has garnered nominal attention as a potential host for the production of biofuel. There was a lack of available genetic tools to alter molecular pathways in Clostridium pasteurianum mainly due to difficulties in genetic
CHAPTER 5
accessibility of the organism This is largely due to the current inability to transfer DNA to C. pasteurianum, in addition to lack of a genome sequence for this organism. Based on early genetic studies, it appears efforts were in place to conduct genetic manipulation of C. pasteurianum, since a method for producing and regenerating protoplasts was developed (Clarke et al., 1979) and a Type-II restriction endonuclease was identified as a potential barrier to gene transfer (Richards et al., 1988). In contrast to Gram-negative bacteria, Gram-positive cells possess an extensive exterior network of peptidoglycan which physically restricts passage of exogenous DNA into the cell. For this reason, electrotransformation of Gram-positive species is generally less efficient than Gram- negative strains (Trevors et al., 1992). Poor electrotransformation efficiency of Gram- positive bacteria is further compounded within the clostridia due to the unusually high production of non-specific cell-wall-associated nucleases (Mermelstein et al., 1992). A number of highly-specific clostridial Type-II restriction endonucleases have also been identified (Jennert et al., 2000; Mermelstein et al., 1992; Klapatch et al., 1996), including CpaAI from C. pasteurianum ATCC 6013 (Richards et al., 1988), highlighting the importance of DNA protection via methylation of the transforming DNA. Unidentified restriction-modification systems are likely the underlying cause of electrotransformation recalcitrance that has been observed with certain species, such as C. butyricum (Gozalez- Pajuelo et al., 2005). But recently several recent strategies have been employed in an attempt to alter the central metabolism of C. pasteurianum to enhance its productivity. It is clear that metabolic engineering will play a central role in the development of C.
pasteurianum as an efficient industrial producer. To this end, an electroporation- mediated method of transformation was recently established (Pyne et al., 2013), allowing gene transfer to C. pasteurianum. Such efficient plasmid transfer paves the way to
rational metabolic engineering strategies, including gene disruption, knockdown, and overexpression techniques (Papoutsakis et al., 2008; Pyne et al., 2014), very few of which have been explored using C. pasteurianum (Pyne et al., 2015; Schwarz et al., 2017).
5.1.1 hydA- Iron only hydrogenase
Hydrogenase is the key enzyme involved in catalyzing the reversible reaction of protons to molecular hydrogen (H2). Hydrogenase was first described by Stephenson and Stickland in 1931 and since then extensive research has been conducted on the structure, function and mechanism of hydrogenase. Hydrogenases are characterized by the metal atoms present in their active site which plays an important role in the enzyme activity and based on this hydrogenases can be categorized into three distinct classes as [NiFe], [Fe-Fe] and [Fe] hydrogenase. Among these, [NiFe] constitute the largest number of hydrogenases found in bacteria and archaea. It has a bimetallic active site with nickel and an iron atom. [NiFe] hydrogenases are membrane bound enzymes, characterized by a dual function of H2 formation and consumption refered to as uptake hydrogenases widely found in cyanobacteria such as Nostoc and Anabena (Kim et al., 2011). Unlike the second group, [FeFe]-hydrogenases are monomeric and contain only one catalytic subunit with a two iron atoms, is found in both eukaryotes and prokaryotes. This group of hydrogenase is particularly involved in H2 production lacking H2 consumption activity. And lastly the [Fe]-hydrogenases or [Fe]-only-hydrogenases which are only found in methanogenic Archaea (Calusinska et al., 2010). It catalyzes CO2 reduction with H2 to methane (Vignais and Colbeau, 2004). In bacteria, hydrogenases regulate the H+ levels in the cells and thus, are actively involved in redox regulation and in
CHAPTER 5
maintaining the proton motive force. Clostridia rely foremost on [FeFe]-hydrogenases but a few species possess [Fe]-hydrogenase and [NiFe]-hydrogenase (Calusinska et al., 2010). In C. pasteurianum, 4 hydrogenases were found in the genome (CLPA_c00280, CLPA_c07060-70, CLPA_c33960 and CLPA_c37830) (Poehlein et al., 2015). With CLPA_c07060 and CLPA_c07070 C. pasteurianum seems to possess a rare [NiFe]- hydrogenase with the genes encoding the small and large subunit, respectively. The BLAST searches and the reports by Pyne et al. (2014) affirms the respective gene of CLPA c00280 as hydA. Further knock-down of hydA by Schwarz et al., 2017 clearly show that deletion of the hydrogenase encoded by CLPA c00280 is tightly involved with the fermentative pathway in C. pasteurianum. Metabolic engineering strategies has been applied to hydrogenases mostly for increasing solvent production or decreasing other by- product formation such as acids. Many reports are focused on knock-down or deletion of hydrogenase which involves hydA knock-down via anti-sense RNA in C. pasteurianum (Pyne et al., 2015), attempted disruption of C. acetobutylicum hydA (Cooksley et al., 2012), deletion of the hydrogenase maturase gene hydG in C. thermocellum (Biswas et al., 2015). Previous work for improvement of hydrogen generation in clostridia involves over-expression of iron-only hydrogenase gene (hydA) in C. paraputrificum (Morimoto et al., 2005, Klein et al., 2010). Over-expression of hydrogenase in C. acetobutylicum DSM 729 did not show any influence on fermentation pattern and it was concluded that intracellular hydrogenase concentration does not limit hydrogen formation in this strain (Klein et al., 2010). Therefore the role of hydA as the key enzyme in hydrogen formation varies from organism to organism. However in C. pasteurianum the key role of hydA in the fermentative pathway was identified by Schwarz et al., 2017. Therefore, attempts to overexpress the hydA were carried out in this study.
To my knowledge there is no report of hydA over-expression in C. pasteurianum in terms of increasing hydrogen yield. This study presents the fermentation pattern of C.
pasteurianum with overexpressed hydA.
5.1.2 dhaD1:dhaK- Glycerol dehydrogenase and dihydroxyacetone kinase
Under anerobic conditions bioconversion of glycerol is mediated through the dha system which is composed of four enzymes viz., glycerol dehydrogenase, dihydroxyacetone kinase, glycerol dehydratase and 1, 3-propanediol oxidoreductase (Forage et al., 1982a, 1982b).These enzymes are involved in two parallel pathways of anaerobic micro-organisms namely, oxidative and reductive pathway. The oxidative pathwayleads from glycerol to pyruvate to enter the fermentative pathway. The second pathway is reductive and goes from glycerol via 3-hydroxypropionaldehyde (3-HPA) to 1,3-PDO (Biebl et al., 1999; Malaviya et al., 2012).
In the oxidative branch glycerol dehydrogenase (dhaD or gldA) converts glycerol to dihydroxyacetone using NAD+ and dihydroxyacetone is phosphorylated by dihydroxyacetone kinase (dhaK) in a coupled reaction with pyruvate kinase to dihydroxyacetone-phosphate which enters glycolysis and fermentation. This metabolic branch consumes glycerol and produces by-products such as ethanol, butanol, acetic acid, butyric acid, hydrogen, succinic acid and also provides energy and NADH to drive the reductive pathway for 1,3-propanediol formation.
Metabolic flux analysis showed that the glycolytic flux during anaerobic fermentation of glycerol was exclusively controlled by glycerol dehydrogenase and dihydroxyacetone kinase in Escherichia coli (Cintolesi et al., 2012).Various efforts to engineer glycerol catabolism and downstream metabolic pathways for production of
CHAPTER 5
various value-added products, including fuels, chemicals, and biomaterials have been reported in the literature. Glycerol dehydrogenase and dihydroxyacetone kinase are the significant enzymes that play key role in glycerol uptake pathway. These enzymes has been metabolically explored in microorganisms particularly for fermentative production of 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), and 2,3-butanediol (2,3- BDO) from glycerol (Wong et al., 2014; Yang et al., 2013; Jung et al., 2011; Clomburg et al., 2011; Maervoet et al., 2016). Overexpression of dhaD or gldA has resulted in improved yield of 1,2-propanediol in E.coli and S. cerevisiae (Jung et al., 2011;
Clomburg et al., 2011). Yazdani and Gonzalez showed that simultaneous overexpression of dhaKLM and gldA increased glycerol utilization and ethanol synthesis (Yazdani and Gonzalez, 2008). Glycerol consumption is partially strengthened by overexpression of the glycerol uptake genes: glycerol dehydrogenase (gldA) and dihydroxyacetone kinase (dhaKLM) in E. coli which enhanced ethanol yield under microaerobic condition (Wong et al., 2014). Moreover, it has been reported that there are two genes encoding glycerol dehydrogenase in Klebsiella pneumoniae viz., dhaD and gldA (Wang et al., 2014).
Similarly, in C. pasteurianum glycerol dehydrogenase is encoded by two genes dhaD1 and dhaD2 which was depicted from the complete genome of C. pasteurianum. In this study we have cloned the dhaD1 gene
In this study, we have used crude glycerol synthesized in laboratory scale from soybean oil (Sarma et al., 2016) as the substrate for H2 production. Our previous study on metabolic flux analysis (Sarma et al., 2017) explained that doubling the glycerol uptake flux channeled metabolic flux towards pyruvate node thereby increasing the flux towards efficient generation of hydrogen. This could be initiated by overexpression of the immediate genes of glycerol utilization pathway viz., dhaD and dhaK. The genes for
glycerol dehydrogenase (dhaD), dihydroxyacetone kinase (dhaK), glycerol dehydratase, and 1,3-propanediol oxidoreductase (dhaT) are encoded in one and the same regulon named dha (Forage and Lin, 1982; Tong et al., 1991). To my knowledge there is no report on over-expression of dhaD1 and dhaK in C. pasteurianum in terms of increasing hydrogen yield. This study presents the fermentation pattern of C. pasteurianum with overexpressed dhaD1-dhaK.
In the work presented here, we have engineered C. pasteurianum by overexpression of combination of key genes dhaD1, dhaK and hydA for enhanced glycerol utilization and hydrogen production respectively. This attempt was made based on our previous studies on metabolic flux analysis of C. pasteurianum with glycerol as the substrate (Sarma et al., 2017) which proposed two theoretical approaches viz., doubling the glycerol uptake rate and elimination of butyrate pathway enhances the flux towards H2.
5.2 MATERIALS AND METHODS